For detection of analytes present in biological samples, biosensors having a large variety of detection systems have been proposed. Of known biosensors, those utilizing surface plasmon resonance (SPR) are sensitive to changes in refractive index at surfaces and in the vicinities thereof of thin metal films (e.g., see A. Szabo, et al., Curr. Opin. Strnct. Biol. 5(1995) 699–705). SPR enables an in situ observation of procedures occurring between a surface and complex biological solutions that allows, e.g., acquisition of data from analytes in real time without requiring tagging of the analytes. It, therefore, is suitable for obtaining both kinetic and thermodynamic parameters, and hence SPR sensor is one of those biosensors which are drawing attention.
As a typical biosensor chip having this kind of surface, BIACORE® which is commercially available from Amersham Pharamacia Biotech., Inc. can be named, which is provided in form of a chip in which a translucent matrix of dextran with carboxylated ends is immobilized on a thin gold film. A patent which is considered to claim such a detection surface is Japanese Patent No. 2815120 (corresponding to U.S. Pat. No. 5,242,828 and EP 0 589 867B1) Gazette. This Official Gazette describes a surface formed by the steps of linking organic molecules expressed by a formula HS—R—Y (wherein R stands for a hydrocarbon chain having a chain length exceeding ten atoms and which may be interrupted with hetero atom(s), and Y stands for a ligand or an active group for covalently bonding a biocompatible porous matrix thereto) onto a membrane surface of the free-electron metal such as gold, silver or the like via the thiol (or mercapto) groups therein, whereby covering said surface with a close-packed monolayer of said organic molecules, and thereafter covalently bonding to the surface a hydrogel as said biocompatible porous matrix, said hydrogel comprising agarose, dextran, polyethylene glycol and the like which may have functional group(s) for linking the ligand.
Japanese Patent No. 3071823 (corres. to U.S. Pat. No. 5,763,191 and EP 0 574 000B1) describes a surface formed of a spacer molecule (C1–C30 alkylene chain) which links onto a support member via a sulfur atom (of mercapto group) and to which covalently bonded are, by order, a hydrophilic linker (a straight chain molecule of 4 to 15 atoms in chain length) and a solid phase reactant (biotin derivative residue). The same patent also describes a compound expressed by the following formula, as a typical biotinylated compound which forms such a surface:

The molecular chains on said surface which have solid phase reactant may further be diluted with diluting molecules which do not have the solid phase reactant (i.e. in the above formula for example, biotin derivative residue), or which have neither the solid phase reactant nor hydrophilic linker.
Roberts et al., J. Am. Chem. Soc., 1998, 120, 6548–6555 describes formation of self-assembled mono-molecular layer (SAM) on a golden surface via mercapto groups, using a compound based on HS-spacer molecule (C11 alkylene chain)-hydrophilic linker (a chain formed of 3 or 6 ethylene oxide units). This document also teaches that a surface formed of a mixture of a compound whose hydrophilic linker portion consists of 3 ethylene oxide units and a compound whose hydrophilic linker portion consists of 6 ethylene oxide units (an oligopeptide ligand binding to its end) promotes cells' ligand-specific binding but reduces accumulation of proteins by so bound cells. Holmlin et al., Langmuir. 17, 2841–2850 (2001) also discusses suppression of protein adsorption onto a surface containing said SAM and ampholytic ion.
Pavey et al., Biomaterials, 20 (1999) 885–890 discloses a surface onto whose SPR detecting thin metal film various combinations of two kinds of poly(ethylene oxide)n-poly(propylene oxide)m-poly(ethylene oxide)n triblock copolymers are adhered, with a suggestion that on so formed surfaces poly(ethylene oxide) chains would extend in the solution to form a brush-like architecture. Furthermore, the document shows: on the surfaces onto which two kinds of triblock copolymers of different poly-(ethylene oxide) chain length (n) are adhered, generally protein (bovine serum albumin) adsorption is less, compared with the surfaces to which one kind only of said triblock copolymer is adhered.
Among the foregoing prior art documents, Japanese Patent No. 2815120 discloses that a monolayer surface in which organic molecules are densely packed can be obtained by chemical adsorption of an organic compound whose chain (R) length exceeds 10 atoms, preferably 12–30 atoms, e.g., 16-mercaptohexadecanol having hydrophobic, considerably large alkylene chain, onto a metal surface via thiol group. So obtained monolayer exhibits storage stability, and the patent furthermore suggests it also can be an effective barrier layer to protect the metal surface from chemical corrosion. Onto such a barrier layer a hydrogel which minimizes protein compatibility and non-specific interaction is bound. Hence aforesaid BIACORE® (carrying hydrogel of dextran) which likely is a preferred embodiment of said patented invention has been reduced to practice. It is, however, by no means easy to have the barrier layer uniformly carry the hydrogel and precise operations are required. Also although non-specific adsorption of protein is considerably reduced, there is still room for further improvement.
Where biotinyl (solid phase reactant) on the surface formed of aforesaid biotinylated compound is densely present, said Japanese Patent 3071823 dared to sparsely bind the biotinylated compound onto the surface of a support material, or link to the surface both said diluent molecules and molecules having biotynyl residue (solid phase reactant), by using the diluent molecules having no biotynyl residue and the corresponding molecules having biotinyl residue at a ratio of 10:1–2:1, for improving slow binding of, for example, biotin and free avidin which forms non-covalently bonded pair with biotin. Such a surface or that proposed by Roberts et al. have hydrophilic linker portion formed of up to about 5 to 6 ethylene oxide units but do not have a hydrogel layer like the one in Japanese Patent 2815120, and hence may cause non-specific adsorption of impurity proteins other than the object protein (e.g., streptavidin) or cells.
Pavey et al. adheres said triblock copolymers onto a metal surface via their hydrophobic blocks, i.e., poly(propylene oxide) domain, and it is difficult to obtain a surface with stability, uniformity and reproducibility, like ordinary polymer coating (cf. U.S. Pat. No. 4,415,666). Moreover, it is also difficult to raise density of poly-(ethylene oxide) chains.
A part of the present inventors discovered, as a surface from which such shortcomings of those prior art surfaces as above described were removed or reduced, a surface prepared by the steps of dissolving a polymer formed mainly of poly(ethylene oxide) (which may hereafter be abbreviated as PEG) having mercapto (—SH) group at one end and the other end being optionally protected, in a buffer solution, and contacting said solution with golden surface of an SPR sensor chip for about an hour. They found that non-specific protein adsorption onto said surface could be reduced at least to the level equivalent to that onto the commercial BIACORE® sensor chip surface CM5, and have filed a patent application directed to such a surface (cf. WO 01/86301).
According to said Japanese Patent 2815120, the R in said HS—R—Y chains must be a hydrocarbon group having at least 10 atoms, to enable to closely pack the metal surface with said chains. Whereas, according to the Japanese Patent 3071823, a biotinylated compound having a hydrophilic linker of a chain length, for example, of 4–15 atoms (1–3 of ethylene oxide units) is used to forms a surface on which chains of said compound are sparsely linked. Surprisingly, however, according to said WO 01/86301, the inventors thereof discovered macromolecules which are entirely different form those polymers described in U.S. Pat. No. 3,071,823 or Roberts et al. in that the former comprises such long chain polymers as that their PEG domain has a molecular weight of 1,000–10,000, could be effectively linked to the metallic surface via their mercapto groups, in an aqueous solution. That is, when such a polymer as described in WO 01/86301 is used, the hydrophilic PEO chains and proteins in the solution cause spatial repulsion, and it was a common recognition among skilled artisans that a PEO layer which was hydrated to reduce such an interaction at the surfaces was flexible and mobile. Nevertheless, the desired amount (or at an adequate density) of said PEO chains are found to be stably linked to the surface.
Whereas, for the surface as described in WO 01/86301, further reduction in non-specific adsorption of impurity proteins thereon is desirable if all possible, similarly for BIACORE® sensor surface. Therefore, the present invention aims at provision of a surface which enables further reduction in non-specific adsorption thereonto, compared with the surface described in WO 01/86301.